The research proposed here is concerned with the organization and regulation of two aminoacyl-tRNA synthetases and the tRNAs cognate to one of them. The genes for the Alpha and Beta subunits of phenylalanyl-tRNA synthetase, theonyl-tRNA synthetase, and translation initiation factor IF3 are clustered closely together; in this research the transcription and regulation of the genes will be analyzed. Promoters will be identified by means of an abortive initiation reaction of RNA polymerase. Transcripts synthetized in vitro with purified RNA polymerase will be identified and localized on the physical and genetic map. DNA sequences will be determined, particularly in regulatory regions. The 5 feet ends of the RNA transcripts will be sequenced, so that the start sites can be located exactly in the DNA sequence. In vivo transcription will be studied by mapping with S1 nuclease. A second project concerns the family of tRNAs specific for threonine. Previous work in this laboratory has shown that there are four genes, three coding for the three major tRNA species plus a fourth more minor gene with apparent regulatory significance. The proposed research will characterize this latter gene further. The specific approaches to be used include: (1) molecular cloning of the gene; (2) localization on a physical map; (3) hybridization tests for the presence of other tRNA genes nearby; (4) DNA sequence analysis; and (5) in vitro regulatory studies. The long-term objective of this research is to elucidate how the cell regulates the activity of tRNA and synthetase genes, which are among the most essential in any organism because of their crucial role in protein synthesis. The health-relatedness of such basic research is indirect, but the information gained may aid in the understanding of diseases caused by aberrations in cellular regulation.